Fig. 5

Localized knockdown of fibronectin in the uterine horns of mice decreased the number of implantation sites, the number of embryos successfully conceived, and the expression of CD31 and CK8. A, Treatment process of the mice is shown. The male and female mice were mated. The day when a vaginal plug was observed was designated day 1 (D1) of pregnancy. Before embryo implantation (morning of D4), the in vivo-jetPEI/si-Fn1 complex or in vivo-jetPEI/si-Ctrl complex was injected as shown in (A). Then, the number of uterine implantation sites was counted on D6 of pregnancy (D, n = 5 mice/group), and uterine tissues were collected for PCR and Western blot to verify the knockdown efficiency of fibronectin (B and C, n = 3-6 mice/group). On D8, the number of successfully implanted embryos was counted (E, n = 5 mice/group), and the uterine sections were stained for CD31 and CK8. The average optical density (AOD) of the IHC staining intensity of CD31 was calculated by dividing integral optical density (IOD) by area. Fluorescence intensity of CK8 was quantified by dividing IntDen of CK8 by IntDen of DAPI. All images were quantitatively measured using ImageJ software (F, n = 4-6 mice/group). Black arrowheads indicate implantation sites; red arrowheads indicate resorption sites; E, embryo; asterisk, absence of embryo; DB, decidua basalis. The results are presented as the mean ± SEM from at least three independent experiments. P values were calculated by two-tailed Student’s t test or Mann–Whitney U test for the summarized quantitative results. Groups without letters in common are significantly different from each other (P < 0.05). P values of primary interest are denoted as * < 0.05, ** < 0.01, and *** < 0.001